Polypeptide inhibitors of SMAD3 polypeptide activities

ABSTRACT

This document provides polypeptide inhibitors of Smad3 polypeptide activities. For example, methods and materials for using polypeptides (e.g., polypeptides designed to include both a cell penetrating amino acid sequence and an amino acid segment of a SH3 domain of a SNX9 polypeptide) to inhibit one or more Smad3 polypeptide activities are provided. This document also provides methods and materials for using RNA interference to treat a disease (e.g., a fibrotic disease) in a mammal (e.g., a human).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/426,455, filed Feb. 7, 2017 (now U.S. Pat. No. 10,144,929), whichclaims the benefit of U.S. Provisional Application Ser. No. 62/354,447,filed Jun. 24, 2016, U.S. Provisional Application Ser. No. 62/297,277,filed Feb. 19, 2016, and U.S. Provisional Application Ser. No.62/295,843, filed Feb. 16, 2016. The disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GM055816 andGM054200, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND 1. Technical Field

This document relates to polypeptide inhibitors of Smad3 polypeptideactivities. For example, this document provides methods and materialsfor using polypeptides (e.g., polypeptides designed to include both acell penetrating amino acid sequence and an amino acid segment of a SH3domain of a SNX9 polypeptide) to inhibit one or more Smad3 polypeptideactivities. This document also relates to methods and materials forusing RNA interference to treat a disease (e.g., a fibrotic disease) ina mammal (e.g., a human).

2. Background Information

Transforming growth factor beta (TGFβ) is a 25 kDa polypeptide thatregulates a variety of cellular processes including matrix deposition,mitosis, development, differentiation, and apoptosis. The primaryintracellular mediators of TGFβ action are the Smad proteins, althoughnon-Smad pathways have been reported, often in a cell-type specificcontext. Three general categories of Smad proteins were identified:receptor-regulated Smads (R-Smads; Smads2 and 3 for TGFβ or Activin andSmads1, 5, and 8 for BMPs); common-mediator Smad (Co-Smad; Smad4); andinhibitory Smads (I-Smads; Smads6 and 7). The R- and Co-Smad proteinsshuttle continuously between the nucleus and cytoplasm in unstimulatedcells as well as in the presence of TGFβ.

SUMMARY

This document provides polypeptide inhibitors of Smad3 polypeptideactivities. For example, this document provides methods and materialsfor using polypeptides (e.g., polypeptides designed to include both acell penetrating amino acid sequence and an amino acid segment of a SH3domain of a SNX9 polypeptide) to inhibit one or more Smad3 polypeptideactivities.

This document also provides methods and materials for using RNAinterference to treat a disease (e.g., a fibrotic disease) in a mammal(e.g., a human). For example, small interfering RNA (siRNA) or shorthairpin RNA (shRNA) can be designed to target SNX9 nucleic acid andtrigger RNA interference against SNX9 nucleic acid expression.Administration of such siRNA or shRNA (or compositions containing orconfigured to express such siRNAs or shRNAs) can result in a reducedlevel of SNX9 polypeptide expression within a mammal. In some cases,siRNA or shRNA designed to target SNX9 nucleic acid and trigger RNAinterference against SNX9 nucleic acid expression (or compositionscontaining or configured to express such siRNAs or shRNAs) can be usedto treat a disease such as carpal tunnel syndrome, lung, kidney, and/orliver fibrosis, glomerulosclerosis, cirrhosis, vascular restenosis,radiation-induced fibrosis, multiple sclerosis, traumatic brain injury,proliferative vitreoretinopathy, ocular capsule injury, or scleroderma.

In general, one aspect of this document features a polypeptidecomprising, or consisting essentially of, a cell penetrating amino acidsequence and an amino acid segment of a SH3 domain of a SNX9polypeptide, wherein the amino acid segment is less than 45 amino acidresidues (e.g., from about 15 to about 40 amino acid residues) inlength. The cell penetrating amino acid sequence can be an amino acidsequence set forth in Table 1. The amino acid segment can be an aminoacid sequence set forth in Table 2. The polypeptide can comprise anamino acid sequence set forth in Table 3.

In another aspect, this document features a nucleic acid moleculeencoding a polypeptide comprising, or consisting essentially of, a cellpenetrating amino acid sequence and an amino acid segment of a SH3domain of a SNX9 polypeptide, wherein the amino acid segment is lessthan 45 amino acid residues (e.g., from about 15 to about 40 amino acidresidues) in length. The cell penetrating amino acid sequence can be anamino acid sequence set forth in Table 1. The amino acid segment can bean amino acid sequence set forth in Table 2. The polypeptide cancomprise an amino acid sequence set forth in Table 3.

In another aspect, this document features a host cell comprising anucleic acid molecule encoding a polypeptide comprising, or consistingessentially of, a cell penetrating amino acid sequence and an amino acidsegment of a SH3 domain of a SNX9 polypeptide, wherein the amino acidsegment is less than 45 amino acid residues (e.g., from about 15 toabout 40 amino acid residues) in length. The cell penetrating amino acidsequence can be an amino acid sequence set forth in Table 1. The aminoacid segment can be an amino acid sequence set forth in Table 2. Thepolypeptide can comprise an amino acid sequence set forth in Table 3.

In another aspect, this document features a method for treating fibrosisin a mammal. The method comprises, or consists essentially of,administering a composition comprising an siRNA or shRNA molecule, or anucleic acid encoding the siRNA or shRNA molecule, to the mammal,wherein the siRNA or shRNA molecule targets SNX9 nucleic acid andtriggers RNA interference against expression of the SNX9 nucleic acid,and wherein the severity of the fibrosis is reduced following theadministering step. The mammal can be a human. The composition cancomprise the siRNA molecule. The composition can comprise the shRNAmolecule. The composition can comprise nucleic acid encoding the siRNAmolecule. The composition can comprise nucleic acid encoding the shRNAmolecule. The nucleic acid can be a viral vector. The SNX9 nucleic acidcan be human SNX9 nucleic acid. The severity of the fibrosis can bereduced by at least 25 percent following the administering step. Theseverity of the fibrosis can be reduced by at least 50 percent followingthe administering step. The severity of the fibrosis can be reduced byat least 75 percent following the administering step. The fibrosis canbe lung fibrosis. The fibrosis can be liver fibrosis. The fibrosis canbe kidney fibrosis.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1C. SH3 domain of sorting nexin 9 (SNX9) specifically bindspSmad3 and prevents nuclear import. (A) Cartoon depicting domains infull length (FL) SNX9 and constructs used for GST pull down assays.Lysates from AKR-2B cells untreated (−) or stimulated (+) for 45 minuteswith 5 ng/mL TGFβ were incubated with GST beads or the indicated fusionpolypeptides immobilized on GST beads. Bound polypeptides were elutedand assessed by Western analysis for pSmad3 or pSmad2. Cell lysatereflects signal obtained from 10 μg total protein. (B) AKR-2B cells weretransduced for 90 minutes with the indicated concentrations of theTAT-SNX9(SH3) fusion polypeptides. Following washing and 1 hour TGFβ (5ng/mL) treatment, nuclear fractions were isolated, and Western blottedfor pSmad2, pSmad3, or histone deacetylase 1 (HDAC) (Left). Quantitationof nuclear pSmads was performed with Image J software and represents themean+/−sd of two experiments (Right). (C) AKR-2B cells were transducedas in (B) with TAT-SH3 or TAT-LC (1.8 μM) and treated+/−TGFβ (5 ng/mL)for 1 hour. Immunofluorescence for Smad3 or the HA-tagged TAT-SNX9(SH3)fusion polypeptide was performed, and nuclei were stained with DAPI(Left panels). Quantitation of nuclear Smad3 from 30 cells in each oftwo experiments (Right).

FIGS. 2A-2C. TAT-SH3 inhibits Smad3-dependent responses. (A) AKR-2Bcells were transiently transfected with a Smad3 (3TP), Smad2 (ARE), orSmad1/5/8 (BRE) reporter construct, and luciferase activity determinedfollowing 12 hours incubation in the absence (−) or presence (+) of theindicated ligand (5 ng/mL TGFβ; 10 ng/mL BMP4) or TAT-SNX9(SH3) fusionpolypeptide (1.5 μM). Data represent the mean+/−SEM of threeexperiments. (B) RT-PCR analysis of Smad3 (PAI-1 and CTGF) and Smad2(MMP2) responsive gene following 24 hour treatment of AKR-2B cells withthe indicated concentration of TAT-SNX9(SH3) fusion polypeptide or 5ng/mL TGFβ. Loading was verified by GAPDH expression. (C) Soft agarcolony formation was performed following seven days growth in thepresence (+) or absence (−) of TGFβ (10 ng/mL) or the indicatedTAT-SNX9(SH3) fusion polypeptide (1.5 μM). Data reflects the mean+/−sdof triplicate wells from three experiments.

FIGS. 3A-3E. Inhibition of Smad3 signaling by a defined region of theSH3 domain in SNX9. (A) Cartoon depicting constructs used for His pulldown assays. AKR-2B lysates were incubated with the indicatedTAT-SNX9(SH3) fusion polypeptide and immunoblotted for bound pSmad3 ortotal TAT-SNX9(SH3) fusion polypeptide (His). (B) Nuclear fractions wereprepared and assessed as in FIG. 1B following transduction with theindicated TAT-SNX9(SH3) fusion polypeptide (Left). Quantitation(mean+/−sd) of nuclear pSmad2 or pSmad3 from 2 experiments (Right). (C)Immunofluorescence of nuclear Smad3 was determined as in FIG. 1C from 30cells in each of two experiments in the absence (−) or presence (+) of 5ng/mL TGFβ and the indicated TAT-SNX9(SH3) fusion polypeptide (1.5 μM).(D) qPCR of Smad3 (PAI-1 and CTGF) and Smad2 (Goosecoid and Furin)responsive genes following 24 hours in the absence (−) or presence (+)of TGFβ (5 ng/mL), SB431542 (10 μM; TβRI inhibitor), or the indicatedTAT-SNX9(SH3) fusion polypeptide. Data reflect mean+/−sd from threeexperiments. (E) TGFβ (10 ng/mL) stimulated soft agar colony formationin the absence or presence of the indicated TAT-SNX9(SH3) fusionpolypeptide (1.5 μM) or SB431542 (10 μM). Data reflects the mean+/−sd oftriplicate wells from three experiments.

FIGS. 4A-4E. A point mutant of TAT-SH3-2 abolishes the inhibitory actionon Smad3 responses. (A) Schematic depicting TAT-SNX9(SH3) fusionpolypeptide constructs (Left). * in SH3-2M (mutant) reflects G to Vmutations at amino acids 36-38. His pull down of pSmad3 bound toTAT-SNX9(SH3) fusion polypeptides was performed as in FIG. 3A (Middle).AKR-2B cells were transduced for 90 minutes with 1.5 μM of TAT-SH3-2,TAT-SH3-2M, or TAT-LC. Western analysis was performed for the indicatedproteins following 24 hour treatment in the absence (−) or presence (+)of TGFβ (5 ng/mL) or SB431542 (10 μM) (Right). (B) qPCR of Smad3 (CTGFand Smad7) and Smad2 (Goosecoid and MixL) responsive genes as in FIG.3D. Data reflect mean+/−sd from three experiments. (C) Scratch assayswere performed on AKR-2B cells following transductions with theindicated TAT-SNX9(SH3) fusion polypeptides and are representative ofthree separate experiments. Red bands indicate the leading edgefollowing 24 hours in the absence (Control) or presence of 5 ng/mL TGFβ.(D) Soft agar colony formation as in FIG. 3E. Data reflects themean+/−sd of triplicate wells from three experiments. (E) AKR-2B cellswere transiently transfected with a BMP (BRE), EGF (SRE), or PDGF(MMP-1) reporter construct and luciferase activity determined following12 hour incubation in the absence (−) or presence (+) of the indicatedligand (10 ng/mL BMP4, EGF, or PDGF), inhibitor (10 μM Dorsomophin; 3 μMLapatinib; 2 μM CP868) or TAT-SNX9(SH3) fusion polypeptide (1.5 μM).Data represent the mean+/−SEM of n=3 for BRE and n=2 for SRE and MMP-1.

FIGS. 5A-5D. Bleomycin (BLM)-induced lung remodeling is attenuated byTAT-SH3-2. (A) Hematoxylin and Eosin (H&E), Masson's Trichrome (MT), orfibronectin (anti-fibronectin and Hematoxylin) staining ofrepresentative paraffin-embedded lung sections from Control (salinetreated) or mice challenged with BLM for 28 days and treated daily with0.5 mg/kg TAT-SH3-2 or TAT-SH3-2M beginning 14 days following initialBLM insult (×8). (B) qPCR of the indicated genes (Coll, collagen 1;CTGF, connective tissue growth factor; FN, fibronectin) in murine lungtissue harvested on day 28 from mice challenged with BLM (+) or saline(−) and treated daily with vehicle (−; methocel/saline) or the indicatedamount (μg) of TAT-SH3-2 or TAT-SH3-2M as in (A). Data reflect mean+/−sdof n=4. (C) Mice were treated with saline or BLM as in (A) and on day 14administered daily vehicle (−) or the indicated concentration of TATpeptide. Animals were sacrificed on day 28, and hydroxyproline contentwas determined as described herein. Data reflect mean+/−sd of n=3.*P<0.005, **P<0.002. (D) Mice were treated as in (C), and qPCR of theindicated genes (Col Iα1 (collagen Iα1); CTGF (connective tissue growthfactor); and FN (fibronectin)) were assessed in lung tissue harvested onday 28. Data reflect mean+/−sd of n=5. *P<0.005, **P<0.001.

FIGS. 6A-6B. (A) Treatment with TAT-SH-2 stabilizes lung gas exchange inBLM-challenged mice. Time-dependent fluctuation of oxygen saturation(SpO₂) levels (determined on room air) in mice challenged with BLM (orsaline) for 28 days and treated 1×/day with vehicle (methocel/saline),0.5 mg/kg of TAT-SH3-2 or TAT-SH3-2M beginning 14 days after initial BLMinsult. Error bars reflect SEM from n=4. (B) Time-dependent fluctuationof oxygen saturation (SpO₂) levels (determined on room air) in micechallenged with BLM (or saline) for 28 days and treated 1×/day withvehicle (methocel/saline) or the indicated concentration of TAT-SH3-2 orTAT-SH3-2M beginning 14 days after initial BLM insult. Error barsreflect mean+/−sd from n=5. *P<0.05, **P<0.01.

FIG. 7 contains a schematic of three overlapping polypeptides (SH3-2-1(SEQ ID NO:33); SH3-2-2 (SEQ ID NO:34); and SH3-2-3 (SEQ ID NO:35)) usedto further define the active motif in SNX9 (top). AKR-2B cells weretransduced with the indicated TAT-SNX9(SH3) fusion polypeptides, andnuclear Smad3 staining was determined as in FIG. 1C. Data reflectnuclear Smad3 from 30 cells in each of two experiments (+/−SEM).

FIG. 8 is a graph plotting the expression of CTGF. TGFβ stimulated CTGFgene expression was examined in subsynovial connective tissue as inFIGS. 3D and 4B following addition of TAT-SH3-2 (SNX9 peptide) orTAT-SH3-2M (Control peptide). The results were normalized to controltreatments without TGFβ.

FIG. 9 is a graph plotting the expression of PAI-1 by cells exposed tothe indicated treatments. TGFβ stimulated PAI-1 gene expression wasexamined in subsynovial connective tissue as in FIG. 8.

FIGS. 10A-10B contain graphs showing that TAT polypeptides do notinhibit in vitro cell proliferation. (A) AKR-2B (10% DMEM/FBS) and MRCS(10% EMEM/FBS) cells were seeded at 2.5×10³ or 1×10⁴ cells/96 wellplate, respectively. 24 hours after seeding, the medium was removed andreplaced with DMEM or EMEM containing vehicle (0.1% DMSO), SH3-2 (1.5μM), or SH3-2M (1.5 μM) either in 10% or 0.1% FBS for 24 hours prior toMTT assay. Absorbance was measured at 570 nm. (B) AKR-2B (1.25×10⁴/well)or MRCS (5×10⁴/well) cells were seeded in 24 well plates for 24 hours.Cultures were treated as in FIG. 10A, and cell counts were determinedfollowing an additional 24 hours and 48 hours of incubation. Resultsrepresent mean±SEM from three independent experiments.

FIGS. 11A-11C. TAT-SH3-2 inhibits profibrotic responses in human lungfibroblasts. (A) Normal human lung fibroblasts (NHLF) or lungfibroblasts from idiopathic pulmonary fibrosis (IPF) patients weretransduced with the indicated TAT polypeptide. TGFβ (5 ng/mL) orSB431542 (10 μM) was then added for an additional 24 hours. Images wereobtained on a LSM510 confocal microscope following F-actin labeling withphalloidin-TRITC and DAPI nuclei staining. Scale bar: 10 μm. (B) Westernanalysis for the indicated polypeptides (α-SMA, alpha smooth muscleactin; CTGF, connective tissue growth factor; and GAPDH, glyceraldehydephosphate dehydrogenase) subsequent to TAT polypeptide transduction and24 hour treatment in the absence (−) or presence (+) of TGFβ (5 ng/mL)or SB431542 (10 μM). (C) NHLF or IPF fibroblasts were treated as in (A),and qPCR was performed as described herein. Results represent mean±SEMfrom three independent experiments. *P<0.05, **P<0.005, *** P<0.001.

FIG. 12. C57BL/6 mice received intratracheal instillation of saline orbleomycin (BLM). On day 14, all animals began daily treatment witheither vehicle (methocel/saline) or 1 mg/kg of TAT-SH3-2. Blood sampleswere obtained at days 0, 14, and 28 from the facial vein ofunanesthetized animals and assessed for effect on the indicated liverenzymes and inflammatory cells. Quantification of lymphocytes,monocytes, and neutrophils were measured using a VetScan HM5 Analyzer.Serum levels (U/L, units per liter; g/dL, grams per deciliter) ofalkaline phosphatase (ALP), alanine aminotransferase (ALT), and albuminwere determined using a Piccolo Xpress Chemistry analyzer. Data arepresented as mean+/−SEM of n=5.

FIGS. 13A-13C. Mice were infected with 1×10⁸ pfu adenovirus particlesexpressing control (GFP) or active TGFβ by tracheal instillation. On day21, all animals began daily treatment with either vehicle(methocel/saline) or 1 mg/kg of the indicated TAT polypeptide. (A) Ondays 21 and 35, peripheral blood oxygen was determined. (B and C) Micewere sacrificed on day 39 and processed for lung hydroxyproline content(B) or qPCR expression of the indicated genes (C) (CTGF, connectivetissue growth factor; α-SMA, alpha smooth muscle actin; Col Iα1,collagen Iα1). For panels A-C, data reflect mean+/−SEM of n=8 and n=16for adenovirus-GFP and adenovirus-TGFβ, respectively. *P<0.05,**P<0.001, ***P<0.0005.

DETAILED DESCRIPTION

This document provides polypeptide inhibitors of Smad3 polypeptideactivities. For example, this document provides methods and materialsfor using polypeptides (e.g., polypeptides designed to include both acell penetrating amino acid sequence and an amino acid segment of a SH3domain of a SNX9 polypeptide) to inhibit one or more Smad3 polypeptideactivities. In some cases, a polypeptide having both a cell penetratingamino acid sequence and an amino acid segment of a SH3 domain of a SNX9polypeptide can be designated a TAT-SNX9(SH3) fusion polypeptide.

Any appropriate cell penetrating amino acid sequence can be used to makea polypeptide described herein. For example, the amino acid sequencesset forth in Table 1 can be used as a cell penetrating amino acidsequence. Other examples include those described elsewhere (e.g.,Kauffman et al., Trends Biochem. Sci., 40:749-64 (2015)).

In some cases, a cell penetrating amino acid sequence can range inlength from about 9 amino acid residues to about 30 amino acid residues(e.g., from about 9 amino acid residues to about 25 amino acid residues,from about 9 amino acid residues to about 20 amino acid residues, fromabout 10 amino acid residues to about 30 amino acid residues, from about10 amino acid residues to about 25 amino acid residues, from about 10amino acid residues to about 20 amino acid residues, or from about 12amino acid residues to about 20 amino acid residues).

TABLE 1 Cell penetrating amino acid sequences. Amino acid sequenceSEQ ID NO: GYGRKKRRQRRR 1 RQIKIWFQNRRMKWKK 2 RRRRRRRRR 3AGYLLGKINLKALAALAKKIL 4 PLIYLRLLRGQF 5

Any appropriate amino acid segment of a SH3 domain of a SNX9 polypeptidecan be used to make a polypeptide described herein. For example, anamino acid sequence set forth in Table 2 can be used as an amino acidsegment of a SH3 domain of a SNX9 polypeptide to make a polypeptideinhibitor of Smad3 polypeptide activities. In some cases, an amino acidsegment of a SH3 domain of a SNX9 polypeptide can range in length fromabout 12 amino acid residues to about 60 amino acid residues (e.g., fromabout 15 amino acid residues to about 60 amino acid residues, from about20 amino acid residues to about 60 amino acid residues, from about 25amino acid residues to about 60 amino acid residues, from about 12 aminoacid residues to about 50 amino acid residues, from about 12 amino acidresidues to about 45 amino acid residues, from about 12 amino acidresidues to about 40 amino acid residues, from about 12 amino acidresidues to about 35 amino acid residues, from about 15 amino acidresidues to about 45 amino acid residues, from about 15 amino acidresidues to about 30 amino acid residues, or from about 15 amino acidresidues to about 20 amino acid residues).

TABLE 2 Amino acid segments of a SH3 domain of a SNX9 polypeptide.Amino acid sequence SEQ ID NO: IITITNPDVGGGWLEG 6TVNEGEIITITNPDVGGGWLEGRNIKGERGL 7 MATKARVMYDFAAEPGNNELTVNEGEIITIT 8NPDVGGGWLEGRNIKGERGLVPTDYVEILPS

In some cases, a polypeptide inhibitor of Smad3 polypeptide activitiescan have an amino acid sequence as set forth in Table 3. In some cases,a polypeptide inhibitor of Smad3 polypeptide activities described hereincan range in length from about 20 amino acid residues to about 100 aminoacid residues (e.g., from about 30 amino acid residues to about 100amino acid residues, from about 40 amino acid residues to about 100amino acid residues, from about 20 amino acid residues to about 90 aminoacid residues, from about 20 amino acid residues to about 75 amino acidresidues, from about 20 amino acid residues to about 50 amino acidresidues, from about 30 amino acid residues to about 90 amino acidresidues, from about 25 amino acid residues to about 65 amino acidresidues, from about 30 amino acid residues to about 60 amino acidresidues, from about 35 amino acid residues to about 55 amino acidresidues, or from about 40 amino acid residues to about 50 amino acidresidues).

TABLE 3 Amino acid sequences of exemplary polypeptide inhibitors of Smad3polypeptide activities. Amino acid sequence SEQ ID NO:

 9 GRNIKGERGL

10 VNEGEIITITNPDVGGGWLEGRNIKGERGLVPTDYVEILPS

11 GYGRKKRRQRRRGSMATKARVMYDFAAEPGNNELTVNEGEIITITNPDVG 12GGWLEGRNIKGERGLVPTDYVEILPS GYGRKKRRQRRRGSTVNEGEIITITNPDVGGGWLEGRNIKGERGL13 GYGRKKRRQRRRGSIITITNPDVGGGWLEG 14RQIKIWFQNRRMKWKKMATKARVMYDFAAEPGNNELTVNEGEIITITNPD 36VGGGWLEGRNIKGERGLVPTDYVEILPSRQIKIWFQNRRMKWKKTVNEGEIITITNPDVGGGWLEGRNIKGERGL 15RQIKIWFQNRRMKWKKIITITNPDVGGGWLEG 16RRRRRRRRRMATKARVMYDFAAEPGNNELTVNEGEIITITNPDVGGGWLE 17GRNIKGERGLVPTDYVEILPS RRRRRRRRRTVNEGEIITITNPDVGGGWLEGRNIKGERGL 18RRRRRRRRRIITITNPDVGGGWLEG 19AGYLLGKINLKALAALAKKILMATKARVMYDFAAEPGNNELTVNEGEIIT 20ITNPDVGGGWLEGRNIKGERGLVPTDYVEILPSAGYLLGKINLKALAALAKKILTVNEGEIITITNPDVGGGWLEGRNIKGER 21 GLAGYLLGKINLKALAALAKKILIITITNPDVGGGWLEG 22PLIYLRLLRGQFMATKARVMYDFAAEPGNNELTVNEGEIITITNPDVGGG 23WLEGRNIKGERGLVPTDYVEILPS PLIYLRLLRGQFTVNEGEIITITNPDVGGGWLEGRNIKGERGL 24PLIYLRLLRGQFIITITNPDVGGGWLEG 25 Single underline = junction; doubleunderline = HA tag amino acid sequence

A polypeptide inhibitor of Smad3 polypeptide activities can have one ormore amino acid substitutions (e.g., one, two, three, four, five, six,seven, or more) relative to an amino acid sequences set forth in Tables1-3. Amino acid substitutions can be made, in some cases, by selectingsubstitutions that do not differ significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. For example, amino acidresidues can be divided into groups based on side-chain properties: (1)hydrophobic amino acids (norleucine, methionine, alanine, valine,leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine,serine, and threonine); (3) acidic amino acids (aspartic acid andglutamic acid); (4) basic amino acids (asparagine, glutamine, histidine,lysine, and arginine); (5) amino acids that influence chain orientation(glycine and proline); and (6) aromatic amino acids (tryptophan,tyrosine, and phenylalanine). Substitutions made within these groups canbe considered conservative substitutions. Non-limiting examples ofuseful substitutions include, without limitation, substitution of valinefor alanine, lysine for arginine, glutamine for asparagine, glutamicacid for aspartic acid, serine for cysteine, asparagine for glutamine,aspartic acid for glutamic acid, proline for glycine, arginine forhistidine, leucine for isoleucine, isoleucine for leucine, arginine forlysine, leucine for methionine, leucine for phenyalanine, glycine forproline, threonine for serine, serine for threonine, tyrosine fortryptophan, phenylalanine for tyrosine, and/or leucine for valine.Further examples of conservative substitutions that can be made at anyposition within a polypeptide described herein are set forth in Table 4.

TABLE 4 Examples of conservative amino acid substitutions OriginalPreferred Residue Exemplary substitutions substitutions Ala Val, Leu,Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu CysSer Ser Gln Asn Asn Glu Asp Asp Gly Pro Pro His Asn, Gln, Lys, Arg ArgIle Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val,Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu,Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr TyrTrp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleucine Leu

In some embodiments, a polypeptide provided herein can include one ormore non-conservative substitutions. Non-conservative substitutionstypically entail exchanging a member of one of the classes describedabove for a member of another class. Such production can be desirable toprovide large quantities or alternative embodiments of such compounds.Whether an amino acid change results in a functional polypeptide canreadily be determined by assaying the specific activity of thepolypeptide using, for example, methods disclosed herein.

In some cases, a nucleic acid molecule can be designed to encode apolypeptide described herein. For example, a viral vector can beconstructed to encode a polypeptide having an amino acid sequence setforth in Table 3. Nucleic acid molecules encoding a polypeptidedescribed herein can be identified and isolated using molecular biologytechniques, e.g., as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989).

Vectors containing a nucleic acid encoding a polypeptide describedherein also are provided. A “vector” is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. An “expressionvector” is a vector that includes one or more expression controlsequences, and an “expression control sequence” is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence.

In the expression vectors provided herein, a nucleic acid (e.g., anucleic acid encoding a polypeptide described herein) can be operablylinked to one or more expression control sequences. As used herein,“operably linked” means incorporated into a genetic construct so thatexpression control sequences effectively control expression of a codingsequence of interest. Examples of expression control sequences includepromoters, enhancers, and transcription terminating regions. A promoteris an expression control sequence composed of a region of a DNAmolecule, typically within 100 to 500 nucleotides upstream of the pointat which transcription starts (generally near the initiation site forRNA polymerase II). To bring a coding sequence under the control of apromoter, it is necessary to position the translation initiation site ofthe translational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of expression control sequences in acell when RNA polymerase is able to transcribe the coding sequence intomRNA, which then can be translated into the polypeptide encoded by thecoding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. In somecases, an expression vector such as pTAT-HA, pGEX4T2, or pSF-CMV-Neo canbe used to deliver a polypeptide described herein to a mammal (e.g., ahuman, a rodent such as a mouse or rat, a dog, a cat, a pig, a bovinespecies, or a horse) to be treated. Numerous vectors and expressionsystems are commercially available from such corporations as Novagen(Madison, Wis.), Clonetech (Palo Alto, Calif.), Stratagene (La Jolla,Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

As described herein, a polypeptide containing a cell penetrating aminoacid sequence and an amino acid segment of a SH3 domain of a SNX9polypeptide, or a nucleic acid encoding such a polypeptide, can be usedto inhibit Smad3 polypeptide activities. Examples of Smad3 polypeptideactivities that can be inhibited by a polypeptide described hereininclude, without limitation, phosphorylated Smad3 nuclear import, softagar colony formation, target gene/protein expression,migration/invasion, and lung fibrosis (e.g., lung fibrosis in a murinemodel such as profibrotic target genes and lung function as defined byperipheral blood oxygenation). In some cases, a polypeptide containing acell penetrating amino acid sequence and an amino acid segment of a SH3domain of a SNX9 polypeptide can be used to treat a mammal having adisease such as carpal tunnel syndrome, lung, kidney, and/or liverfibrosis, glomerulosclerosis, cirrhosis, vascular restenosis,radiation-induced fibrosis, multiple sclerosis, traumatic brain injury,proliferative vitreoretinopathy, ocular capsule injury, or scleroderma.Examples of mammals that can be treated as described herein include,without limitation, humans, rodents (e.g., mice or rats), rabbits,simian species, ovine species, porcine species, bovine species, caninespecies, horses, or cats.

Any appropriate method can be used to formulate a polypeptide containinga cell penetrating amino acid sequence and an amino acid segment of aSH3 domain of a SNX9 polypeptide, or a nucleic acid encoding such apolypeptide, into a therapeutic composition. In addition, anyappropriate method can be used administer such a therapeutic compositionto a mammal as described herein. Dosages typically are dependent on theresponsiveness of the mammal to the therapeutic composition, with thecourse of treatment lasting from several days to several months, oruntil a suitable response is achieved. Optimum dosages can varydepending on the relative potency of a therapeutic composition, andgenerally can be estimated based on those levels found to be effectivein in vitro and/or in vivo animal models. Therapeutic compositionsprovided herein may be given once or more daily, weekly, monthly, oreven less often, or can be administered continuously for a period oftime (e.g., hours, days, or weeks).

The polypeptides, or nucleic acids, can be admixed, encapsulated,conjugated or otherwise associated with other molecules, molecularstructures, or mixtures of compounds such as, for example, liposomes,receptor or cell targeted molecules, or oral, topical or otherformulations for assisting in uptake, distribution and/or absorption.

This document also provides methods and materials for using RNAinterference to treat a disease (e.g., a fibrotic disease) in a mammal(e.g., a human). For example, siRNA or shRNA can be designed to targetSNX9 nucleic acid and trigger RNA interference against SNX9 nucleic acidexpression. In some cases, a human SNX9 nucleic acid sequence can beused to design an siRNA or an shRNA that targets SNX9 nucleic acid andtriggers RNA interference against SNX9 nucleic acid expression. A humanSNX9 nucleic acid can be as set forth in Genbank Accession No. NM 016224(GI No. 525313625). Examples of siRNA molecules that can be used totrigger RNA interference against human SNX9 nucleic acid expressioninclude, without limitation, GCUGCUGAACCUGGAAAUA (SEQ ID NO:26),GGUUCCCACAGACUACGUU (SEQ ID NO:27), CCAAAGAAAGAUCUCCAUU (SEQ ID NO:28),GCACUCACAAGGGAGCAAU (SEQ ID NO:29), AACAGUCGUGCUAGUUCCUCA (SEQ ID NO:30;Soulet et al., Mol. Biol. Cell., 16(4):2058-2067 (2005)),UAAGCACUUUGACUGGUUAUU (SEQ ID NO:31; Bendris et al., Mol. Biol. Cell.,27(9):1409-1419 (2016)), and GGGACUUUGUAGAGAAUUU (SEQ ID NO:32; Bendriset al., Mol. Biol. Cell., 27(9):1409-1419 (2016)).

Any appropriate method can be used to design an siRNA or an shRNA thattargets SNX9 nucleic acid and triggers RNA interference against SNX9nucleic acid expression. For example, software programs such as thosedescribed elsewhere (see, e.g., Naito et al., Nucleic Acids Res., 32(WebServer issue):W124-W129 (2004)) can be used to design an siRNA or anshRNA that targets SNX9 nucleic acid (e.g., human SNX9 nucleic acid) andtriggers RNA interference against SNX9 nucleic acid expression (e.g.,human SNX9 nucleic acid expression).

Once designed, a particular siRNA or shRNA can be assessed in vitro orin vivo to confirm its ability to trigger RNA interference against SNX9nucleic acid expression (e.g., human SNX9 nucleic acid expression). Forexample, a particular siRNA or shRNA can be administered to a mammal,and the level of SNX9 nucleic acid or SNX9 polypeptide expression withinthe mammal (or particular tissues or cells of the mammal) can beassessed before and after administration to identify those siRNA orshRNA molecules having the ability to trigger RNA interference againstSNX9 nucleic acid expression. In some cases, the methods and materialsdescribed elsewhere (e.g., Soulet et al., Mol. Biol. Cell.,16(4):2058-2067 (2005), or Bendris et al., Mol. Biol. Cell.,27(9):1409-1419 (2016)) can be used to confirm that a particular siRNAor shRNA has the ability to trigger RNA interference against SNX9nucleic acid expression.

Any appropriate method can be used to deliver one or more siRNA or shRNAmolecules provided herein to cells or tissue within a mammal such asthose described elsewhere (e.g., Kanasty et al., Nature Materials,12(11):967-977 (2013) or Xu et al., Asian Journal of PharmaceuticalSciences, 10(1):1-12 (2015)). For example, siRNA or shRNA having theability to trigger RNA interference against SNX9 nucleic acid expressioncan be configured into lipid nanoparticles such as those describedelsewhere (e.g., U.S. Patent Application Publication No. 2011/0224447)to deliver the siRNA or shRNA to cells within a mammal (e.g., a human).In some cases, one or more siRNA and/or shRNA molecules having theability to trigger RNA interference against SNX9 nucleic acid expressionprovided herein can be delivered to liver cells within a mammal totreat, for example, liver fibrosis. For example, delivery vehiclescontaining N-acetyl-d-galactosamine such as those described elsewhere(e.g., Dhande et al., Biomacromolecules, 17(3):830-840 (2016)) can beused to deliver one or more siRNA and/or shRNA molecules having theability to trigger RNA interference against SNX9 nucleic acid expressionto cells (e.g., liver cells). In some cases, siRNA conjugated withα-tochopherol using techniques such as those described elsewhere (e.g.,Murakami et al., Scientific Report, 5:17035 (2015)) can be used todeliver one or more siRNA and/or shRNA molecules having the ability totrigger RNA interference against SNX9 nucleic acid expression to cells(e.g., liver cells). In some cases, cyclodextrin compositions such asthose described elsewhere (e.g., Arima et al., Curr. Top. Med. Chem.,14(4):465-77 (2014)) can be used to deliver one or more siRNA and/orshRNA molecules having the ability to trigger RNA interference againstSNX9 nucleic acid expression to cells. In some cases, a biodegradablepolymeric matrix such as those described elsewhere (e.g., Ramot et al.,Toxicol Pathol., May 4 (2016) or Golan et al., Oncotarget.,6(27):24560-70 (2015)) can be used to deliver one or more siRNA and/orshRNA molecules having the ability to trigger RNA interference againstSNX9 nucleic acid expression to cells. In some cases, aerosolformulations of siRNA such as those described elsewhere (e.g., Lam etal., Adv. Drug. Deliv. Rev., 64(1):1-15 (2012)) can be used forpulmonary delivery of one or more siRNA and/or shRNA molecules havingthe ability to trigger RNA interference against SNX9 nucleic acidexpression to cells.

As described herein, a composition can be formulated to contain one ormore siRNA and/or shRNA molecules having the ability to trigger RNAinterference against SNX9 nucleic acid expression (e.g., a compositioncan be formulated to contain one or more siRNA and/or shRNA moleculeshaving the ability to trigger RNA interference against SNX9 nucleic acidexpression in combination with a deliver vehicle such as a lipidnanoparticle, N-acetyl-d-galactosamine, cyclodextrin, and/orbiodegradable polymeric matrix such as those described above). Such acomposition containing one or more siRNA and/or shRNA molecules havingthe ability to trigger RNA interference against SNX9 nucleic acidexpression can be administered to a mammal to treat a disease. Examplesof diseases that can be treated with a composition containing one ormore siRNA and/or shRNA molecules having the ability to trigger RNAinterference against SNX9 nucleic acid expression include, withoutlimitation, carpal tunnel syndrome, lung, kidney, and/or liver fibrosis,glomerulosclerosis, cirrhosis, vascular restenosis, radiation-inducedfibrosis, multiple sclerosis, traumatic brain injury, proliferativevitreoretinopathy, ocular capsule injury, and scleroderma. In somecases, a composition containing one or more siRNA and/or shRNA moleculeshaving the ability to trigger RNA interference against SNX9 nucleic acidexpression can be administered to a mammal to treat a fibrotic disease(e.g., lung, kidney, and/or liver fibrosis).

In some cases, a nucleic acid molecule can be designed to express ansiRNA and/or shRNA molecule having the ability to trigger RNAinterference against SNX9 nucleic acid expression. For example, a viralvector can be constructed to encode an siRNA and/or shRNA moleculehaving the ability to trigger RNA interference against SNX9 nucleic acidexpression.

In the expression vectors provided herein, a nucleic acid (e.g., anucleic acid encoding an siRNA and/or shRNA molecule having the abilityto trigger RNA interference against SNX9 nucleic acid expression) can beoperably linked to one or more expression control sequences. As usedherein, “operably linked” means incorporated into a genetic construct sothat expression control sequences effectively control expression of acoding sequence of interest. Examples of expression control sequencesinclude promoters, enhancers, and transcription terminating regions. Apromoter is an expression control sequence composed of a region of a DNAmolecule, typically within 100 to 500 nucleotides upstream of the pointat which transcription starts (generally near the initiation site forRNA polymerase II).

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. In somecases, an expression vector such as pTAT-HA, pGEX4T2, or pSF-CMV-Neo canbe used to deliver an siRNA and/or shRNA molecule described herein to amammal (e.g., a human, a rodent such as a mouse or rat, a dog, a cat, apig, a bovine species, or a horse) to be treated. Numerous vectors andexpression systems are commercially available from such corporations asNovagen (Madison, Wis.), Clonetech (Palo Alto, Calif.), Stratagene (LaJolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).

As described herein, an siRNA and/or shRNA molecule described herein, ora nucleic acid encoding such an siRNA and/or shRNA molecule describedherein, can be used to inhibit Smad3 polypeptide activities. Examples ofSmad3 polypeptide activities that can be inhibited by an siRNA and/orshRNA molecule having the ability to trigger RNA interference againstSNX9 nucleic acid expression include, without limitation, phosphorylatedSmad3 nuclear import, soft agar colony formation, target gene/proteinexpression, migration/invasion, and lung fibrosis (e.g., lung fibrosisin a murine model such as profibrotic target genes and lung function asdefined by peripheral blood oxygenation). In some cases, an siRNA and/orshRNA molecule having the ability to trigger RNA interference againstSNX9 nucleic acid expression can be used to treat a mammal having adisease such as carpal tunnel syndrome, lung, kidney, and/or liverfibrosis, glomerulosclerosis, cirrhosis, vascular restenosis,radiation-induced fibrosis, multiple sclerosis, traumatic brain injury,proliferative vitreoretinopathy, ocular capsule injury, or scleroderma.Examples of mammals that can be treated using an siRNA and/or shRNAmolecule having the ability to trigger RNA interference against SNX9nucleic acid expression as described herein include, without limitation,humans, rodents (e.g., mice or rats), rabbits, simian species, ovinespecies, porcine species, bovine species, canine species, horses, orcats.

Any appropriate method can be used to formulate an siRNA and/or shRNAmolecule having the ability to trigger RNA interference against SNX9nucleic acid expression, or a nucleic acid encoding such an siRNA and/orshRNA molecule, into a therapeutic composition. In addition, anyappropriate method can be used administer such a therapeutic compositionto a mammal as described herein. Dosages typically are dependent on theresponsiveness of the mammal to the therapeutic composition, with thecourse of treatment lasting from several days to several months, oruntil a suitable response is achieved. Optimum dosages can varydepending on the relative potency of a therapeutic composition, andgenerally can be estimated based on those levels found to be effectivein in vitro and/or in vivo animal models. Therapeutic compositionsprovided herein may be given once or more daily, weekly, monthly, oreven less often, or can be administered continuously for a period oftime (e.g., hours, days, or weeks).

An siRNA and/or shRNA molecule described herein, or nucleic acidencoding an siRNA and/or shRNA molecule described herein, can beadmixed, encapsulated, conjugated or otherwise associated with othermolecules, molecular structures, or mixtures of compounds such as, forexample, liposomes, receptor or cell targeted molecules, or oral,topical or other formulations for assisting in uptake, distributionand/or absorption.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Inhibition of Profibrotic Smad3 Action by CellPenetrating Peptides that Block Smad3 Nuclear Import

Cell Culture

AKR-2B cells were grown in DMEM supplemented with 10% fetal bovine serum(FBS). Prior to use, 2.5×10⁵ cells were seeded on 6 well plates andcultured in 10% FBS/DMEM. The next day, the medium was replaced with0.5% FBS/DMEM, and cells were transduced for 90 minutes with theindicated TAT-containing polypeptide. After transduction, cells wereincubated with or without TGFβ (5 ng/mL) for the indicated times in 0.5%FBS/DMEM.

Generation of TAT-SNX9(SH3) Fusion Polypeptides

TAT-SNX9(SH3) fusion polypeptides were prepared from BL21(DE3)pLysS E.coli (OD600 of 0.4) using techniques similar to those describedelsewhere (Wilkes et al., Mol. Biol. Cell., 26(21):3879-91 (2015)).Briefly, following addition of isopropyl β-d-thiogalactopyranoside to afinal concentration of 0.5 mM and 4 hours induction at 37° C., lysateswere prepared, and the precleared supernatant poured into a TALON MetalAffinity Resin column (Clontech, Mountain View, Calif.). Polypeptideswere eluted (50 mM sodium phosphate, 300 NaCl, and 150 mM imidazole,pH7.4) and dialyzed against PBS using a Slide-A Lyzer Mini Dialysis unit(Thermo Scientific, Rockford, Ill.).

Soft Agar Assay

Soft agar assays were performed as described elsewhere (Rahimi et al.,Cancer Res., 69(1):84-93 (2009)). Briefly, 1×10⁴ cells in 10% FBS/DMEMwere seeded in a 6 well plate in the presence or absence of 10 ng/mLTGFβ (R&D Systems, Minneapolis, Minn.) and the indicated TAT-SNX9(SH3)fusion polypeptides. Following 7 days growth at 37° C., the number ofcolonies>100 μm in diameter were counted using an Optronix Gelcount™(Oxford Optronics, Milton, Abingdon, UK). The results wererepresentative of three separate experiments, each done in triplicate.

Luciferase Reporter Assays

For luciferase assays, 2×10⁵ cells were plated in six well platescontaining 10% FBS/DMEM. The next day, cells were transfected with 2(3TP, BRE, SRE, and MMP1) or 1.5 (ARE; plus 1.5 μg FAST1) μg of theindicated luciferase constructs together with 0.5 μg cytomegalovirus(CMV)-β-galactosidase with TransIT-2020 reagent (Minis Bio, Madison,Wis., USA). Following 24 hour incubation at 37° C., the medium waschanged to 0.5% FBS/DMEM, and the cultures were treated with theindicated TAT-SNX9(SH3) fusion polypeptides for 90 minutes. Aftertransduction, cultures were incubated in the presence or absence of 5ng/mL TGFβ for 12 hours in 0.5% FBS/DMEM. Cells were harvested in 200 μLof reporter lysis buffer (Promega, Madison, Wis.), and luciferaseactivity was determined in a Berthold Lumat 9507 luminometer afternormalization for transfection efficiency with β-galactosidase.

Immunofluorescent Microscopy

2×10⁴ cells were plated onto coverslips in 10% FBS/DMEM and incubatedovernight at 37° C. Cultures were placed in 0.5% FBS/DMEM and transducedwith the indicated TAT-SNX9(SH3) fusion polypeptides for 90 minutes.After transduction and addition of TGFβ (5 ng/mL) for 1 hour, cells wereprocessed as described elsewhere (Wilkes et al., Mol. Biol. Cell.,26(21):3879-91 (2015)). Smad3 was detected using AF488 secondaryantibodies (green), while the HA tag of the TAT-SNX9(SH3) fusionpolypeptides was visualized with AF-594 (red) secondary antibodies, bothfrom Invitrogen (Carlsbad, Calif.). Fluorescence images were collectedon a LSM510 confocal microscope (Carl Zeiss Microimage Inc. Thornwood,N.Y.). Both AF488 and AF594 were secondary antibodies. The primaryantibodies were those to Smad3 and HA, respectively.

Quantitative Reverse Transcription (RT)-PCR Analysis

Following TGFβ stimulation, total RNA was isolated using Rneasy plusMini kit (QIAGEN, Valencia, Calif.), and 1 μg reverse transcribed withthe SuperScript® III Reverse Transcriptase system (Invitrogen, CA).Complementary DNAs were subjected to qPCR with platinum SYBR green qPCRsuperMix-UDG (Invitrogen, CA) or TaqMan gene expression analysis (ThermoScientific, Rockford, Ill.), and sample induction was normalized toHistone H3.

Western Blotting

Cells were lysed for 30 minutes on ice in RIPA buffer (50 mM Tris, pH7.4, 1% Triton X100, 0.25% Sodium deoxycholate, 150 mM NaCl, 1 mM EDTA,pH 8, and 10 mM NaF) containing protease inhibitor cocktail tablets(Roche, Indianapolis). Insoluble material was removed by centrifugationat 18,000×g for 10 minutes, and 10-25 μg of protein was separated by 10%SDS-PAGE. Phospho-Smad2 and -Smad3 specific antibodies were generated,while anti-GAPDH and anti-His tag antibodies were obtained from EMDMillipore (Darmstadt, Germany). Anti-PAI-1, -CTGF, and -histonedeacetylase 1 (HDAC1) antibodies were obtained from Santa CruzBiotechnology (Santa Cruz, Calif.) and Cell Signaling (Danvers, Mass.).

GST and his Pull-Down

Fusion polypeptides containing GST- or His-tags were purified usingGlutathione Superflow or TALON Metal Affinity Resin, respectfully,following the manufacturer's instructions (Clontech, Mountain View,Calif.). To assess SNX9 binding to GST or His constructs, cells wereincubated in the presence or absence of 5 ng/mL TGFβ for 45 minutes.Following RIPA buffer lysis, 500 μg of protein was precleared with GSTResin or TALON Metal Affinity Resin for 2 hours at 4° C. Precleared celllysates were then treated with 5 μg purified GST- or His-fusion proteinsand incubated overnight at 4° C. with gentle shaking. Following additionof GST or TALON Metal Affinity Resin and 2 hours of incubation, thepelleted (3,000×g; 10 minutes) resin was washed several times with PBS,and the remaining proteins were eluted using 1× Laemmli sample buffer.Western blotting was performed as described above.

Isolation of Nuclear Fractions

Nuclear extracts were prepared using NE-PER Nuclear and CytoplasmicExtraction Kits for Cultured Cells (Thermo Scientific, Rockford, Ill.)with the addition of protease inhibitor (Roche, Indianapolis, Ind.) tothe lysis buffers. Following removal of the cytoplasmic extract, thenuclear pellet was washed three times in PBS containing proteaseinhibitor before nuclear lysis and Western analysis.

Animal Models of Pulmonary Fibrosis

Female 18-20 g C57 black mice (Charles River Laboratories) wereadministered bleomycin (BLM; 0.075 U diluted in 50 μL 0.9% normalsaline) or 50 μL 0.9% normal saline alone by tracheal instillation usingan intratracheal aerosolized (Penn-century, Wyndmoor, Pa.) as describedelsewhere (Andrianifahanana et al., FASEB J., 27(11):4444-54 (2013)). Atthis time, animals were shaved around the collar region to allowmonitoring of dissolved oxygen levels (every 3rd day) on room air usinga MouseOX collar clip monitoring system (Starr Life Science Corp.Oakmont, Pa.). TAT-SNX9(SH3) fusion polypeptides were solubilized withDMSO and prepared by thoroughly blending with Methocel (Sigma, Louis,Mo.) at a ratio of 1:7. Mice were treated daily by intraperitonealinjection (100 μL) of a TAT-SNX9(SH3) fusion polypeptide or an equalvolume of methocel/saline beginning on day 14. On day 28, mice wereeuthanized, lungs were dissected, and samples were prepared forimmunohistochemistry and other analyses as described elsewhere(Andrianifahanana et al., FASEB J., 27(11):4444-54 (2013); Daniels etal., J. Clin. Invest., 114:1308-16 (2004); and Wang et al., FASEB J.,19:1-11 (2005)).

Hydroxyproline Assay

The Hydroxyproline Assay Kit from Sigma (St. Louis, Mo.) was used toassess total lung collagen levels. Briefly, following sacrifice lungtissue was washed in PBS, and 30 mg homogenized in 300 μL water. Onehundred μL samples were hydrolyzed in 12 M HCl, and duplicate 50 μLsamples were analyzed according to the manufacturer's recommendations.

Results

A TAT-Containing Sorting Nexin 9 Polypeptide Specifically Blocks pSmad3Nuclear Import and Profibrotic TGFβ Signaling

As described elsewhere, SNX9 has an obligate role in mediatingprofibrotic TGFβ signaling dependent upon phosphorylated (p) Smad3(Wilkes et al., Mol. Biol. Cell., 26(21):3879-91 (2015)). In order toinvestigate whether pSmad3 bound to a defined region(s) in SNX9, GSTfusion constructs encoding either the amino (i.e., SH3 and lowcomplexity (LC) domains) or carboxyl (i.e., Phox and BAR domains) halfof SNX9 were generated, and pull down assays for pSmad3 were performed(FIG. 1A depicts SNX9 domain structure). While elements within the Phoxand BAR domains were unable to bind pSmad3, equivalent pSmad3 bindingwas observed with GST full length (FL) SNX9 and the amino terminalpeptide. To define this interaction further, two overlapping aminoterminal fragments were generated. As shown in FIG. 1A, while equivalentpSmad3 binding was observed with constructs expressing FL SNX9 or thefirst 74 amino acids encoding the SH3 domain, the LC domain exhibitedonly minimal binding. Of note, no pSmad2 association was observed forany of the constructs, consistent with the results described elsewhere(Wilkes et al., Mol. Biol. Cell., 26(21):3879-91 (2015)).

As also described elsewhere, SNX9 was found to be required for thenuclear import of pSmad3, but not pSmad2 (Wilkes et al., Mol. Biol.Cell., 26(21):3879-91 (2015)). Since the SNX9 SH3 domain was capable ofsimilarly binding pSmad3 as the FL protein (FIG. 1A), the following wasperformed to determine whether this was sufficient to inhibit pSmad3nuclear uptake. Constructs were prepared expressing either the SNX9 SH3or LC domains fused to a cell penetrating TAT polypeptide from HIV(Becker-Hapak et al., Methods, 24(3):247-56 (2001); and Rizzuti et al.,Drug Discov. Today, 20(1):76-85 (2015)). Subsequent to TAT polypeptidetransduction, cultures were treated with TGFβ, and nuclear accumulationof pSmad2 or pSmad3 was determined. As shown in FIG. 2B, while aTAT-SNX9(SH3) fusion polypeptide containing the full length SH3 domain(amino acids 1 to 62; designated TAT-SH3 herein) inhibited nuclearimport of pSmad3 in a dose-dependent manner, it had no effect on pSmad2.Furthermore, consistent with the inability of the LC domain to bindR-Smads (FIG. 1A), it was similarly ineffective in modulating nucleartranslocation (FIG. 1B). These biochemical findings were confirmed usingimmunofluorescence (FIG. 1C).

While the results of FIG. 1 demonstrate that a TAT-SNX9(SH3) fusionpolypeptide containing a full length SH3 domain can be capable ofinhibiting the nuclear accumulation of pSmad3 following TGFβ addition,these results did not assess the functional impact of this response. Assuch, to investigate whether this loss was sufficient to inhibitSmad3-mediated responses, the studies shown in FIG. 2 were performed.AKR-2B cells were transfected with luciferase constructs responsive toeither Smad3, Smad2, or bone morphogenetic proteins (BMPs), and theimpact of inhibiting pSmad3 nuclear import by TAT-SH3 determined. WhileSmad3-dependent luciferase activity was inhibited about 70%, nodiscernible effect on either Smad2 or Smad1/5/8 (i.e., BMP) signalingwas observed (FIG. 2A). These luciferase results were extended bothtranscriptionally as well as biologically in FIGS. 2B and 2C,respectively. While Smad3 targets and TGFβ-stimulatedanchorage-independent growth in soft agar (AIG) were inhibited bytransduction with TAT-SH3, induction of the Smad2 regulated MMP-2 genewas unaffected, and the negative control TAT-LC polypeptide was inertfor all responses.

As shown in FIGS. 1-2, the expression of the SH3 domain from SNX9 canfunction in trans as a specific inhibitor of Smad3-regulated responses.The following was performed to define the functional motif(s) in the SH3polypeptide regulating Smad3 signaling and to generate and test, both invitro and in vivo, a mutant SH3 polypeptide unable to bind pSmad3. Toaddress the first of these, three overlapping 25-31 mer TAT-SNX9(SH3)fusion polypeptides were constructed and tested for their ability tobind pSmad3 in cell lysates prepared from TGFβ treated cultures. Asshown in FIGS. 3A-C, TAT-SH3-2 (a TAT-SNX9(SH3) fusion polypeptidehaving SNX9 amino acids 21-51) bound pSmad3 to a similar degree as aTAT-SNX9(SH3) fusion polypeptide having the full length SH3 domain(TAT-SH3) and specifically prevented pSmad3 nuclear import. Moreover,TAT-SH3-2 not only prevented Smad3-dependent transcriptional responsesand AIG, but the inhibition was analogous to that observed with the TβRIkinase inhibitor SB431542 (FIGS. 3D and 3E).

To define the element further, three additional overlapping 15 or 16 merTAT-SNX9(SH3) fusion polypeptides were generated and assessed whetherthey could inhibit pSmad3 nuclear translocation. While activity similarto that obtained with SB431542 was observed with a TAT-containingpolypeptide encoding amino acids 27-42 of SNX9, additional studiesrevealed that the 16 mer did not provide as consistent responses as the31 mer designated TAT-SH3-2. It did, however, suggest that pointmutations in a highly conserved glycine rich region, which waspreviously shown to mediate protein/protein interactions (Harrison etal., J. Biol. Chem., 285(26):20213-23 (2010); Jang and Greenwood,Biochem. Biophys. Res. Commun., 380(3):484-8 (2009); and Shaw et al., J.Biochem., 147(6):885-93 (2010)), might similarly be effective inblocking the inhibitory actions of TAT-SH3-2. This was directly testedin FIG. 4. While TAT-SH3-2 bound pSmad3 and prevented TGFβ induction ofSmad3 targets such as CTGF, PAI-1, and Smad7 to a similar degree asinhibition of TβRI (FIG. 3), point mutations in amino acids 36-38(TAT-SH3-2M) abolished the inhibitory effect (FIGS. 4A and 4B). Neitherpolypeptide impacted the induction of Smad2 targets (FIG. 4B).Furthermore, identical results were observed when Smad3-regulatedbiological responses were examined in the presence of TAT-SH3-2(inhibitory) or TAT-SH3-2M (not inhibitory) (FIGS. 4C and 4D). Last, tofurther confirm the specific action of TAT-SH3-2 on Smad3 action, FIG.4E shows that the luciferase activity stimulated by BMP4, EGF, or PDGFresponsive reporters is unaffected by any of the TAT-SNX9(SH3) fusionpolypeptides.

Cell Penetrating Peptides that Block pSmad3 Action are Effective in aTreatment Model of Pulmonary Fibrosis

As demonstrated herein, a cell penetrating polypeptide encoding adefined region of SNX9 that prevents pSmad3 nuclear import can act intrans to inhibit TGFβ-stimulated biochemical, translational,transcriptional, and biological actions dependent upon pSmad3.Furthermore, the degree of inhibition is analogous to that obtained witha small molecule inhibitor of the TβRI kinase and Smad2-, Smad1/5/8-,EGF-, and/or PDGF-stimulated responses are unaffected. Profibroticactions of TGFβ are primarily mediated via Smad3 (Hoot et al., J. Clin.Invest., 118(8):2722-32 (2008); and Meng et al., J. Am. Soc. Nephrol.,21(9):1477-87 (2010)). A treatment model of bleomycin (BLM)-induced lungfibrosis was used to test, in vivo, the efficacy of intraperitonealadministration of TAT-SH3-2 (or TAT-SH3-2M) commencing 14 days followinginitial BLM insult. As shown in FIGS. 5A and 5C, while TAT-SH3-2improved lung histology, reduced interstitial fibronectin to essentiallybasal levels, and showed a dose-dependent dimunization in total collagenproduction induced by bleomycin, the TAT-SH3-2M peptide unable to bindpSmad3 (FIG. 4A) was ineffective. Consistent with theimmunohistochemistry, qPCR analysis of similarly treated murine lungtissue showed that TAT-SH3-2 polypeptides, but not TAT-SH3-2M,significantly reduced the BLM induction of profibrotic genes includingcollagen 1, connective tissue growth factor, and fibronectin (FIGS. 5Band 5D).

The results provided herein demonstrate that TAT-SNX9(SH3) fusionpolypeptides can prevent pSmad3 nuclear import and can function as aninhibitor of profibrotic TGFβ action both in vitro and in vivo. Thefollowing was performed to determine whether a corresponding improvementin lung physiology also is observed. This was directly examined byassessing the effect of TAT-SH3-2 (and TAT-SH3-2M) on peripheral bloodoxygen saturation (SpO₂). Although an analogous decline in SpO₂ wasobserved in animals receiving the control TAT-SH3-2M polypeptide asthose treated with saline, TAT-SH3-2 stabilized and/or improved gasexchange over the 2-week treatment interval (FIG. 6A). While vehicle orSH3-2M (the inactive TAT-SNX9 peptide) treated animals showed anapproximate 25% decrease in SpO₂ during the treatment regime, adose-dependent improvement in gas exchange was observed with TAT-SH3-2such that animals receiving the highest concentration displayed noadditional loss of lung function (FIG. 6B). Thus, by inhibiting pSmad3action, a parameter of normal lung function was maintained.

Example 2—the Effect of a TAT-SNX9(SH3) Fusion Polypeptide onSubsynovial Connective Tissue Fibrosis in Carpal Tunnel Syndrome (CTS)

Fibroblasts were harvested from CTS patient SSCT tissue (n=3) who hadcarpal tunnel release surgery as described elsewhere (Gingery et al., J.Orthop. Res., 32(11):1444-50 (2014)). The CTS SSCT fibroblasts werecultured in Minimum Essential Media (MEM) supplemented with 10% fetalbovine serum (FBS) and 1% antibiotic/antimycotic. Cultures wereincubated at 37° C. in a humidified, 5% CO₂ atmosphere. 2 million cellsper 6 well plate were cultured overnight. 24 hours later, the media wasaspirated, and the cells were cultured in serum depleted media (0.5%FBS). Cultures were pretreated with TAT-SH3-2 (1.5 μM), TAT-SH3-2M, orvehicle for 90 minutes. Cell cultures were treated with 2 ng/mL TGF-βfor 24 hours.

Total RNA was isolated gene expression was evaluated using quantitativereal-time polymerase chain reaction (qRT-PCR) as described elsewhere(Gingery et al., J. Orthop. Res., 32(11):1444-50 (2014)). TGF-βresponsive genes (connective tissue growth factor (CTGF) and plasminogenactivator inhibitor-1 (PAI-1)) were evaluated with TATA binding proteinas the housekeeping gene.

Statistical analysis of qRT-PCR gene expression was normalized tocontrol, and significance was determined by unpaired Student's t-testfor each gene. The level of statistical significance is set at P<0.05.

Results

CTGF gene expression was down regulated by TAT-SH3-2 compared withvehicle (P<0.05; FIG. 8). No significant differences were observedbetween the three groups with respect to the expression of PAI-1.However, there is a substantial trend toward reduced expression (FIG.9).

These results demonstrate that TAT-SNX9(SH3) fusion polypeptide can beused to attenuate TGF-β mediated gene expression of genes such as CTGFand PAI-1. Targeted blocking of Smad3 signaling as described herein canbe used to treat CTS.

Example 3—RNAi to Treat Lung Fibrosis

A human is identified as having a fibrotic disease such lung fibrosis.Once identified, the human is administered (e.g., intravenously) acomposition having one or more than one siRNA (and/or shRNA) designed totarget SNX9 nucleic acid and trigger RNA interference against SNX9nucleic acid expression. The composition is administered in an amountthat delivers between about 5 μg/kg and 1500 μg/kg of siRNA (or shRNA)to the human. Once administered, the human is monitored to confirm areduction in the severity of the lung fibrosis via imaging (e.g., CTscan) or lung function tests. Repeat doses of the composition areadministered as needed. In such cases, the amount of the subsequentdoses can be lower or higher than the initial dose to achieve a desiredoutcome.

Example 4—RNAi to Treat Liver Fibrosis

A human is identified as having a fibrotic disease such liver fibrosis.Once identified, the human is administered (e.g., intravenously) acomposition having one or more than one siRNA (and/or shRNA) designed totarget SNX9 nucleic acid and trigger RNA interference against SNX9nucleic acid expression. The composition is administered in an amountthat delivers between about 5 μg/kg and 1500 μg/kg of siRNA (or shRNA)to the human. Once administered, the human is monitored to confirm areduction in the severity of the liver fibrosis via biopsy and/ornon-invasive tests (e.g., elastography). Repeat doses of the compositionare administered as needed. In such cases, the amount of the subsequentdoses can be lower or higher than the initial dose to achieve a desiredoutcome.

Example 5—RNAi to Treat Kidney Fibrosis

A human is identified as having a fibrotic disease such kidney fibrosis.Once identified, the human is administered (e.g., intravenously) acomposition having one or more than one siRNA (and/or shRNA) designed totarget SNX9 nucleic acid and trigger RNA interference against SNX9nucleic acid expression. The composition is administered in an amountthat delivers between about 5 μg/kg and 1500 μg/kg of siRNA (or shRNA)to the human. Once administered, the human is monitored to confirm areduction in the severity of the kidney fibrosis via biopsy and/ornon-invasive tests (e.g., elastography). Repeat doses of the compositionare administered as needed. In such cases, the amount of the subsequentdoses can be lower or higher than the initial dose to achieve a desiredoutcome.

Example 6—TAT-SNX9(SH3) Fusion Polypeptides do not Inhibit In Vitro CellProliferation

AKR-2B (10% DMEM/FBS) and MRCS (10% EMEM/FBS) cells were seeded at2.5×10³ or 1×10⁴ cells/96 well plate, respectively. 24 hours afterseeding, the medium was removed and replaced with DMEM or EMEMcontaining vehicle (0.1% DMSO), SH3-2 (1.5 μM), or SH3-2M (1.5 μM)either in 10% or 0.1% FBS for 24 hours prior to MTT assay. Absorbancewas measured at 570 nm. In addition, AKR-2B (1.25×10⁴/well) or MRCS(5×10⁴/well) cells were seeded in 24 well plates for 24 hours. Cultureswere treated with DMEM or EMEM containing vehicle (0.1% DMSO), SH3-2(1.5 μM), or SH3-2M (1.5 μM) either in 10% or 0.1% FBS, and cell countswere determined following an additional 24 hours and 48 hours ofincubation.

Exposure of cells to TAT-SNX9(SH3) fusion polypeptides did not inhibitcell proliferation (FIG. 10).

Example 7—TAT-SH3-2 Inhibits Profibrotic Responses in Human LungFibroblasts

Normal human lung fibroblasts (NHLF) or lung fibroblasts from idiopathicpulmonary fibrosis (IPF) patients were transduced with the indicated TATpolypeptide. TGFβ (5 ng/mL) or SB431542 (10 μM) was then added for anadditional 24 hours. Images were obtained on a LSM510 confocalmicroscope following F-actin labeling with phalloidin-TRITC and DAPInuclei staining. In addition, a Western analysis was performed for α-SMA(alpha smooth muscle actin), CTGF (connective tissue growth factor), andGAPDH (glyceraldehyde phosphate dehydrogenase) subsequent to TATpolypeptide transduction and 24 hour treatment in the absence orpresence of TGFβ (5 ng/mL) or SB431542 (10 μM). Further, qPCR wasperformed using NHLF or IPF fibroblasts similarly treated.

TAT-SH3-2 inhibited profibrotic responses in human lung fibroblasts(FIG. 11).

Example 8—TAT-SH3-2 has No Demonstrable Effect on Murine Liver Enzymesor Inflammatory Cell Recruitment

C57BL/6 mice received intratracheal instillation of saline or bleomycin(BLM). On day 14, all animals began daily treatment with either vehicle(methocel/saline) or 1 mg/kg of TAT-SH3-2. Blood samples were obtainedat days 0, 14, and 28 from the facial vein of unanesthetized animals andassessed for effect on the indicated liver enzymes and inflammatorycells. Quantification of lymphocytes, monocytes, and neutrophils weremeasured using a VetScan HM5 Analyzer. Serum levels (U/L, units perliter; g/dL, grams per deciliter) of alkaline phosphatase (ALP), alanineaminotransferase (ALT), and albumin were determined using a PiccoloXpress Chemistry analyzer.

TAT-SH3-2 exhibited no demonstrable effect on murine liver enzymes orinflammatory cell recruitment (FIG. 12).

Example 9—TAT-SH3-2 Stabilizes Lung Function in Adenovirus-TGFβ Modelsof Pulmonary Fibrosis

Mice were infected with 1×10⁸ pfu adenovirus particles expressingcontrol (GFP) or active TGFβ by tracheal instillation. On day 21, allanimals began daily treatment with either vehicle (methocel/saline) or 1mg/kg of the indicated TAT polypeptide. On days 21 and 35, peripheralblood oxygen was determined. Mice were sacrificed on day 39 andprocessed for lung hydroxyproline content or qPCR expression of CTGF(connective tissue growth factor), α-SMA (alpha smooth muscle actin),and Col Iα1 (collagen Iα1).

TAT-SH3-2 stabilized lung function in adenovirus-TGFβ models ofpulmonary fibrosis (FIG. 13).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A nucleic acid molecule encoding a polypeptidecomprising a cell penetrating amino acid sequence and an amino acidsegment of a SH3 domain of a sorting nexin 9 (SNX9) polypeptide, whereinsaid amino acid segment is less than 45 amino acid residues in lengthand comprises the amino acid sequence set forth in SEQ ID NO:6 or
 7. 2.The nucleic acid molecule of claim 1, wherein said cell penetratingamino acid sequence comprises the amino acid sequence set forth in SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
 3. The nucleic acidmolecule of claim 1, wherein said cell penetrating amino acid sequencecomprises GYGRKKRRQRRRGS (SEQ ID NO:37).
 4. The nucleic acid molecule ofclaim 1, wherein said polypeptide comprises the amino acid sequence setforth in any one of SEQ ID NOs:9, 11, 13-16, 18, 19, 21, 22, 24, and 25.5. The nucleic acid molecule of claim 1, wherein said amino acid segmentcomprises the amino acid sequence set forth in SEQ ID NO:6.
 6. Thenucleic acid molecule of claim 1, wherein said amino acid segmentcomprises the amino acid sequence set forth in SEQ ID NO:7.
 7. A hostcell comprising a nucleic acid molecule encoding a polypeptidecomprising a cell penetrating amino acid sequence and an amino acidsegment of a SH3 domain of a sorting nexin 9 (SNX9) polypeptide, whereinsaid amino acid segment is less than 45 amino acid residues in lengthand comprises the amino acid sequence set forth in SEQ ID NO:6 or
 7. 8.The host cell of claim 7, wherein said cell penetrating amino acidsequence comprises the amino acid sequence set forth in SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
 9. The host cell of claim 7,wherein said cell penetrating amino acid sequence comprisesGYGRKKRRQRRRGS (SEQ ID NO:37).
 10. The host cell of claim 7, whereinsaid polypeptide comprises the amino acid sequence set forth in any oneof SEQ ID NOs:9, 11, 13-16, 18, 19, 21, 22, 24, and
 25. 11. The hostcell of claim 7, wherein said amino acid segment comprises the aminoacid sequence set forth in SEQ ID NO:6.
 12. The host cell of claim 7,wherein said amino acid segment comprises the amino acid sequence setforth in SEQ ID NO:7.